TRANSLOCATION OF BENOMYL IN ELM (Ulmus americana L.)
IX.
SOME ECOLOGICAL CONSEQUENCES OF THE TREATMENTS ON POPULATION
DYNAMICS OF EARTHWORMS
(Lumbrlcus
terrestis L.)
■
by R.
Prasad and R.P.
Moody
-
r
Chemical Control Research
Ottawa,
Institute
Ontario
Information Report CC-X-81
October
1974
"
Translocation of Benomyl in Elm (Ulmus americana L.)
IX.
Some Ecological Consequences of-the Treatments on Population
Dynamics of Earthworms (Lumbrlcus terrestis L.)
!
r
TABLE
ABSTRACT
OF
CONTENTS
(RESUME)
INTRODUCTION
MATERIALS
r
r
and METHODS
(i)
Field Site
(ii)
Earthworms
(iii)
(iv)
(v)
(vi)
2
collection
4
Fungicide Treatment
4
Laboratory Soil Drench
4
Thin Layer Chromatography
(TLC)
Radioactive Labelling
5
5
RESULTS and DISCUSSION
(i)
Field Studies
(ii)
Laboratory Soil Drench
(iii)
Radioactive Experiment
CONCLUSION
■
■
r
r
6
Studies
5
7
9
ACKNOWLEDGEMENTS
H
REFERENCES
12
—
P
1
—
ABSTRACT
Earthworm populations were greatly reduced in forest soil
with benomyl and MBC.
There was 100% mortality of worms,
treated
(Lumbricus terres-
tris L.) held in soil drenched with 100,000 ppm and 10,000 ppm suspensions
of benomyl and MBC after 14 days
exposure in the laboratory.
was higher in the MBC treated soil
LD
than in soil drenched with benomyl.
values for benomyl and MBC were 3000 ppm and 300 ppm,
Thin layer chromatographic
that after 14 days
21.3% MBC,
(TLC)
Mortality rate
analysis of
respectively.
the benomyl drenched soil showed
the fungicide molecule had broken down into
and 0.7% AB.
The
78% benomyl,
Radioactive tracer experiments showed that MBC-C
pi
was most concentrated in
the digestive system of
m
/
treated worms.
*
RESUME
Les populations de vers de terre ont considerablement baisse dans
les sols forestiers traites au benomyl et au MBC.
Apres 14 jours d'exposition
en laboratoire, la mortalite chez les vers (Lumbricus terrestris) gardes dans
un sol sature avec des suspensions de benomyl et de MBC a 100,000 p.p.m.
10,000 p.p.m. a atteint 100 p. 100.
r
et
Le taux de mortalite etait beaucoup plus
eleve dans le sol traite au MBC que dans le sol sature de benomyl.
Les DL
50
pour le benomyl et le MBC ont ete de 3,000 p.p.m. et 300 p.p.m. respectivement.
La chromatographie en couche mince du sol sature de benomyl a montre qu'apres
14 jours, la degradation chimique de la molecule de fongicide avait produit
78 p. 100 de benomyl, 21.3 p.
100 de MBC, et 0.7 p. 100 de AB.
Des dosages
par radio-activite ont montre que la concentration la plus forte en
MBC marque se trouvait dans le systeme digestif des vers
r
(L.
C du
terrestris)
traites.
r
INTRODUCTION
Benomyl, l-(butylcarbamoyl)-2-benzimidazole carbamlc acid, methyl
ester,
is a systemic
fungicide which is presently employed in combating a
wide variety of plant fungal diseases.
demons and Sisler
highly unstable nature of benomyl in aqueous
fungitoxic breakdown product,
(HBC).
suspensions and
2-benzimidazole carbamic
isolated
2
and soil
being done without adequate information on the
highly persistent
3
long
application.
term effects
fungicide in the forest ecosystem.
the
acid, methyl ester
Large quantities of benomyl are being employed in an attempt
control Dutch elm disease both by foliar
r
demonstrated the
to
This
of
is
this
A reduction of mite
population has been observed following benomyl treatment '
.
The inhibitory
effect of benomyl and MBC on earthworm populations has been observed in both
field and laboratory soils
r
r
7
8 9
.
The present paper gives further evidence of
reduction of earthworm populations in treated forest soil,
and describes a
laboratory study of the effect of benomyl and MBC on the earthworm,
Lumbricus
terrestris.
MATERIALS AND METHODS
-
A.
FIELD
STUDIES
(i)
Field Site
Earthworm populations surveys were performed at Shirley's
Ottawa at four sites.
(2,500 ppm).
Plot YM had been sprayed with
These areas contained elm trees
(Ulmus americanus L.)
that had been sprayed during a Dutch elm disease control program.
quadrats
—
near
Plot WB had been sprayed with a benomyl - water
suspension (2,500 ppm) in the spring of 1972.
MBC-C1
Bay,
Three
(2X2') were marked out at the base of each tree as shown in Fig.
Two untreated
areas were selected for controls.
1
r
(ii)
Earthworm Collection
Estimation of earthworm populations in forest soils was carried out
by the method of Raw10.
A solution of formalin (50 ml of 40% formaldehyde/10
litre of water) was poured on each quadrat (2 x 2') of soil and the number of
emerging worms was
Worms
recorded for twenty minutes
(L^
terrestrls) for laboratory use were obtained commercially
from Alves Worm Wholesales, Ottawa,
These were about 15 cm long and held in
captivity for at least two weeks before use.
dried deciduous
(ill)
after application.
leaves
Food consisted of a mixture of
that had been ground in a Wareing blender,
Fungicide Treatment
Benlate,
(50% benomyl W.P.)
used for all fungicide preparations.
obtained from the Dupont Company, was
Benomyl and water suspensions were
prepared without heating and used immediately to prevent decomposition.
MBC-C1 was prepared by heating a benomyl-water suspension with HCl(O.lN)
90° C for one hour.
ff
pH of
The fungicidal formulations (benomyl and MBC-C1) had a
7.
B.
LABORATORY
STUDIES
(i)
Soil Drenching
Worms were held in plastic 2-litre pots
rlO worms per plot.
filled with soil
Fifty ml of each concentration (10,
1000,000 ppm)
to each pot in
subsequent
to
were applied
counted after 3,
each
and contained
Aqueous suspensions of benomyl and MBC-C1 were poured on the
soil surface of each pot.
living worms was
'
at
count.
6,
10,
three
replicates.
100,
1000,
The number of
and 14 days and food was given
10,000,
-
r
r
(ii)
Thin Layer Chromatography
To determine
prepared
(TLC)
composition,
the benomyl and MBC suspensions
for soil drenching were first analysed by TLC following
Siegel and Zabbia
acid
exact
5 -
.
A solution of chloroform,
ethyl acetate,
(1:1:0.4) was employed to develop the TLC plates*.
from the drenched soil,
in
the 100,000 ppm pots,
days when the experiment was
for one hour,
The fate of benomyl
analysed after 14
Cold chloroform was
stirred with
C
the TLC plates.
14
labelled MBC was
Nuclear Corporation,
obtained
California,
from
the International Chemical
with a specific activity of
worms were immersed in an aqueous solution of MBC-C
minutes.
These
14
5 mc/mml.
(725 DPM/ul)
and
Six
for two
treated worms were kept separately in 250 ml Erlenmeyer flasks
for 4 days in the dark at 25 C and 90% humidity.
Then the radioactive
solution used for immersion purposes was mixed into
100 cc of soil in a 3
liter
this beaker containing
soil,
the soil
Radioactive Labelling
P
beaker and six untreated worms were added
drenched with MBC-C
as immersed worms.
r
also
and acetic
and after filtering the solvent was concentrated by evaporation
and spotted on
(Hi)
concluded.
was
the method of
14
to
and kept under the same environmental condition
After h days
the worms were killed with chloroform
vapour and rinsed briefly
under running water
on
Three worms from each treatment type
the exterior surface.
to remove any MBC-C
adhering
(immersed and
soil-drenched) were carefully dissected and separated into digestive system,
reproductive organs,
remaining akin and musculature.
The remaining six worms
were cut into thirds by incision just anterior to the clittelum and by halving
the posterior section.
The components were extracted separately in a series
of hot ethanol-water solutions
*Glass TLC plates
F-254
I
(95%,
70%,
50%,
20%,
0% ethanol).
The extracts
(5 x 20 cm) precoated with 0.25 ml layer of silica Gel
(Brinkman Instruments Inc., Westbury, N.Y.)
and developed to 10 cm.
- 6 -
were concentrated by evaporating and counted in a Picker Nuclear
"
(Ansitron
II), liquid scintillation counter using the appropriate dioxane/toluene
cocktails and the external standards
for correction of quenching and
efficiency of the radioactive samples.
RESULTS AND DISCUSSION
A.
FIELD STUDIES
Following foliar sprays,
I
the fluctuations
in earthworm populations
at Shirley's Bay plots were examined after three months and the results are
shown
in
Table
I.
r
Table
I
Earthworm populations at Shirley's Bay on Treated and Check Areas .
Ave. No.
Treatment
L.
terrestris
Plot WB (Benomyl, 2500 ppm)
Plot YM
(MBC,
of worms/quadrat, mean of 30 replicates
Other
Total
0.03
2.30
2.33
0
1.25
1.25
12.70
15.11
2500 ppm)
Controls (Check)
2.41
Thus it Is quite apparent from the data that the population was
greatly reduced In the sprayed plots and the results are consistent with those
obtained by Stringer and Wright
in apple orchards.
Q
during their examination of the treated soil
In general, worms were more numerous in the benomyl plot
than in the MBC plot and this might be related to the decomposition of
benomyl to MBC.
of
B.
Another interesting feature to note is the acute toxicity
the treatments.to L.
terrestris.
LABORATORY STUDIES ON SOIL DRENCH
The results from this study are presented in Fig. 2 and as can be
-
-
7
-
seen from the graphs it is clear that toxicity is related to the level of
concentration of the fungicides.
As before,the MBC treated pots showed
much greater mortality than benomyl drenched-soil.
The LD _
for the two
fungicides was calculated from the 6th day data and was found to be 3000
ppm and 300 ppm for benomyl and MBC respectively which corroborate
findings that MBC is more toxic to L^,
terrestris than benomyl.
the
(Stringer
and Wright9).
TLC analysis of benomyl drenched soil revealed a partial breakdown
of benomyl into MBC and AB (2-aminobenzimidazole) and the R
r
were 0.62, 0.37, and 0.01,
values obtained
for benomyl, MBC, and AB respectively
These values are similar to those obtained by Siegel and Zabbia
(see Fig.
.
3).
It may be
that MBC was the agent responsible for eliciting mortality, and that benomyl
must first break down into MBC before it becomes toxic to earthworms.
This
would also explain the slower rate of killing in the benomyl drenched-soil.
(iv)
Radioactive Experiment
Further evidence of
14
obtained by feeding of MBC-C
the mechanism and locale of
to live worms.
toxicity was
As can be seen from Table II
the concentration of radioactivity is largely in the digestive tracts of
the
worms.
Table
II
Distribution of radioactivity in various parts of earthworms
after feeding with MBC-C
(DPM/gm fresh material)
Body Farts
Immsersed worms
% Activity
Soil drenched worms
%Activity
Reproductive System
22,241
21.6
20,712
18.7
Digestive System
69,206
67.3
64,587
58.3
Skin and Musculature
11,387
11.1
25,554
23.0
Anterior
Middle
Posterior
BENOMYL
MBC CHLORIDE
L
00
!
10
6
TIME (DAYS)
Fig.
2
u
10
6
3
TIME (DAYSJ
Three dimensional graphs illustrating the effects of concentration of MBC chloride and
benomyl and the period of treatment on mortality rate of earthworms.
-9-
Under both conditions of treatments,the results indicate that
more activity was present in the digestive system than in the body wall.
The activity was however weakest in the anterior section.
It is interesting
to note that the total amount of activity was not appreciably higher in the
1
soil-treated worms which received both external and internal dosage than the
immersed worms.
Stringer and Wright
9
demonstrated the repellent nature of
minute quantities of benomyl on food material and thus lethal effect of
benomyl can result solely from skin contact as opposed to ingestion.
I
experiment did not separate the digestive system into internal
external components.
This
(feces) and
Hence it is not possible to determine whether the
fungicide was transported to the digestive system to be excreted with the
waste products, or was,
in fact, being accumulated in the musculature of
the intestine.
The latter condition would more readily explain mortality.
However,
observed
worms was
it was
that
small probably due
experiment.
to
It was also noted
precipitated evacuation.
activity was
the amount of
feces
the starvation of
that
present in
this
it
seems
actually present in the tissue of
immersed
these worms during
the killing by use of
Considering
the
the
chloroform vapour
more probably
that the
the digestive system.
1
Conclusion
Both field data and laboratory soil-drench experiments confirm
the findings of Stringer and Wright
lethal to earthworms;
MBC is much more toxic
9
Lumbricus terrestris being a very susceptible species.
to worms
lethal effect of benomyl is
due
than benomyl
to
and it
is
possible
the formation of MBC as
down product in the soil environment.
I
that benomyl in minute quantity is
that
a break
the
- 11 -
The radioactive tracer studies reveal that MBC diffuses readily
through the earthworm skin and accumulates within the digestive system.
Further radioisotope studies employing the histoautoradiographic technique
should be carried out to establish the localization of MBC-CU at the
cellular
level.
ACKNOWLEDGMENTS
I
The authors wish to thank Dr. J. J. Fettes, Director, Chemical
Control Research Institute for encouragement and Mr.
technical assistance.
Travnick for
They are indebted to the National Capital Commission
for provision of the forest site.
'
D.
- 12 -
References
m
1.
Clemens, G.P., Sisler, H.D.
2.
Hart, J.H.
3.
Stipes, R.J.
4.
Asari, A., Brandolini, V., Vitali, D.
1972.
Phytopathology.
Plant Disease Reporter.
1972.
Fitopatologiche.
'
1969.
Phytopathology.
59, 705.
56, 685.
63, 735.
1970.
Atti Giornate
61.
5.
Spadofora, R.R., Llndquist, R.K.
6.
Upham,
7.
Kennel, W.Z.
8.
Stringer, A., Wright, M.A.
1973.
Pestic. Sci.
4, 431.
9.
Stringer, A., Wright, M.A.
1973.
Pestic. Sci.
4, 165.
10.
Raw, f., 1959.
11.
Siegel, M.R., Zabbia, A.J.
P.M., Delp, C.J.
1972.
1973.
1972.
J. of Econ. Entom. 65, 1718,
Phytopathology 63, 814.
Pfl Krankl. Pfl. Path. Pfl Shuty.
79, 400.
Nature, London. 184, 1661.
1972.
Phytopathol.
62, 630.